Fluorescence is a property exhibited by certain chemicals that involves the excitation of the electronic state of the molecule through absorption of light of a given energy (wavelength), followed by a return to the ground state via certain electronic processes resulting in the emission of light of lower energy (longer wavelength). This property has been utilized for a variety of applications involving the high sensitivity detection of biological and other kinds of analytes.
One general application of this phenomenon is found in the use of artificial enzyme substrates, prepared by derivatizing a fluorescent compound (fluorophore) with a chemical moiety, to detect the presence of a corresponding enzyme. Successful applications require that the substrate (derivatized fluorophore) have a number of characteristics, including the following:
exhibit little or no background fluorescence from the unreacted substrate under the assay conditions; PA0 be chemically stable under the variety of aqueous environments and pH ranges typically employed for the assay of the enzyme; PA0 release a highly fluorescent product on reaction catalyzed by the target enzyme; PA0 exhibit sufficient turn-over number (number of substrate molecules transformed per second per molecule of enzyme) to permit useful assay times; PA0 have sufficient solubility in aqueous solution to allow zero-order kinetics to be achieved, thus permitting a direct correlation of the rate of reaction to the enzyme concentration; and PA0 exhibit linear kinetics over several orders of magnitude in the concentration of the enzyme (see overview in A. Lehninger, D. Nelson and M. Cox, Principles of Biochemistry, New York, Worth Publishers, 1993). PA0 high relative fluorescence: [.epsilon..times.Q]&gt;10,000, where .epsilon. is the molar absorptivity constant for the fluorophore and Q is the quantum yield (ratio of photons emitted to photons absorbed); PA0 spectral resolution: large Stokes shift (difference between the wavelength of maximum excitation and the wavelength of maximum emission), thus avoiding background noise associated with the excitation beam of light; PA0 photostability (to minimize the process of photo-bleaching); and PA0 ease of coupling and small size (to retain the reactivity of labeled reagents, such as antibodies or oligonucleotides). PA0 excitation wavelength near the output of lasers that have been introduced in recent years, one of the more widely used being the Argon 488 nm laser. Use of lasers, with their narrow-band output, substantially reduces background noise due to the excitation of a variety of other materials that may be present in a biological sample as compared to the use of a full spectrum lamp. Further, the laser's high intensity beam is able to pump more photons into a small volume, thus enhancing the sensitivity of the measurement by maximally exciting all available fluorophores. Both properties contribute to an increase in assay sensitivity; PA0 fluorescent emission at longer wavelengths (&gt;540) to reduce interfering background caused by other sources in the biological sample, including a strong Raman emission for water (605-635 nm), and endogenous fluorescent components in plasma and serum samples that extend up to 600 nm, with a particularly strong emission band from 430-540 nm for serum samples. Hemmila, supra, p. 63-6; PA0 availability of multiple emission wavelengths through adjustments of structures; PA0 presence of a moiety in the fluorophore that causes the level of fluorescence to be indicative of its environment (e.g., pH); PA0 full development of fluorescence under physiological conditions (e.g., pH 6-8, etc.); and PA0 capability to adjust solubility of the fluorophore (low solubility for applications requiring precipitation; high solubility in applications involving homogeneous solution measurement).
One example of the many enzymes that are measured quantitatively for the diagnosis of disease through the use of artificial substrates is alkaline phosphatase (ALP) EC3.1.3.1. Its presence is a valuable indicator of hepatobiliary disease (Mass, "Alkaline Phosphatase Isoenzymes," Clin. Chem. 28:2007-2016, 1982). ALP has also been measured to assess the completeness of milk pasteurization (Rocco, Anal. Chem., 73(6):842-849).
Further, any analyte that can be directly related to the enzyme can also be detected. For example, an enzyme can be chemically conjugated to a polyclonal or monoclonal antibody targeted against a specific biological molecule present in a biological sample. Often the analyte in question is a protein or other chemical that is directly linked to a disease state. The general technique is referred to as Enzyme Immunoassay (EIA). An extensive review of EIA that utilizes fluorescence in particular is presented by Ilkka A. Hemmila in Applications of Fluorescence in Immunoassays, John Wiley & Sons, Inc., New York, 1991.
Several enzymes have found extensive use in fluorescence-based EIA applications, including alkaline phosphatase (R. H. Yolken and P. J. Stopa, J Clin. Microbiol. 10:317, 1979) and beta-D-galactosidase (Hosli et al., Clin. Chem. 24:1325, 1978). The driving force to utilize fluorescence-based EIA is the increase in sensitivity over colorimetric methods, as much as a 1000-fold improvement or more. Ishikawa et al., "Methods for Enzyme-labeling of Antigens, Antibodies, and Their Fragments," in T. T. Ngo, Ed., Non-Isotopic Immunoassay, Plenum Press, New York, 1988.
These same enzymes can also be attached to other molecules, such as DNA and RNA oligonucleotides and used in hybridization-based detection assays in which fluorescence again is generated. An example is the use of a fluorescent substrate (Attophos) to ALP (incorporated via an alkaline phosphatase-streptavidin conjugate) in the detection of Neiseseria gonorrhoeae by a hybridization technique (Cano et al., Eur. J Clin. Microbiol. Infect. Dis. 11(7):602-609, 1992). In another application, alkaline phosphatase is used in the detection of HIV-1 genome by Polymerase Chain Reaction (PCR) techniques, again via an alkaline phosphatase-streptavidin conjugate. (Gerard et al., "Fluorometric detection of HIV-1 genome through use of an internal control, inosine-substituted primers, and microtiter plate format," Clinical Chemistry 42(5):696-703 (1996)).
A second major application of fluorescent compounds is their use in directly labeling a component in a detection system. One important application is a technique known as Fluorescence In Situ Hybridization (FISH). Bauman et al., "A new method for fluorescence microscopical localization of specific DNA sequence by in situ hybridization of fluorochrome-labeled RNA," Exp. Cell Res. 138:485-90, 1980. In this technique a DNA or RNA probe against a specific genetic target is labeled with a fluorophore and is mixed with a sample. The presence of the target is detected when the probe hybridizes with the specific sequence, as revealed by the emission of the attached fluorescent label. Multi-target FISH can be achieved with the use of multiple fluorescent probes (Fox et al., "Fluorescence in situ hybridization: powerful molecular tool for cancer prognosis," Clin. Chem. 41(11):1554-1559, 1995).
Another application for direct-labeling is the use of fluorophore-labeled dideoxynucleotide triphosphates to prepare primers in conjunction with PCR techniques for use in DNA sequencing. In one typical application the fluorophore used was fluorescein (Pastinen et al., "Multiplex, fluorescent, solid-phase minisequencing for efficient screening of DNA sequence variation," Clin. Chem. 42(9):1391-1397, 1996).
The properties of a fluorophore, whether directly incorporated into a detection reagent or employed indirectly via an enzyme substrate, are critical to the achievement of high sensitivity and/or rapid analysis. Some of these properties have been summarized by Hemmila, supra, p. 109. These include:
There are additional properties that add significant value to the utility of a fluorophore(s) in particular applications, such as:
Numerous fluorophores are known, many introduced in recent years, for use in a variety of applications. However, "the development of new fluorescent compounds with desired properties is hampered by the complexity of the fluorescence process. Currently available data are not sufficient to allow any definite generalizations in regard to the relationship between the molecular structure and fluorescence," Hemmila, Applications of Fluorescence in Immunoassays, John Wiley & Sons, Inc. 1991, volume 117, p.109. Guilbault supports Hemmila's conclusions and states "the electronic spectroscopy of large molecules is sufficiently complex that neither empirical generalizations nor more fundamental theoretical arguments are necessarily reliable to enable prediction of the fluorescence characteristics of complex molecules," Guilbault, Practical Fluorescence, Marcel Dekker, Inc., 2d ed., Revised and Expanded, 1990. It is, therefore, not surprising that a review of some representative fluorophores currently available reveal significant shortcomings in one or more of the preferred properties, as shown in the following discussion
4-Methylumbelliferone (4-MU) is one of the oldest fluorophores in use as an enzyme substrate (Yolken and Stopa, supra). Its fluorescent properties include .lambda..sub.EX of 367 nm, .lambda..sub.EM of 449 nm, and a Stokes Shift of 82 nm (.lambda..sub.EX and .lambda..sub.EM represent the wavelength of maximum excitation and emission, respectively). Unfortunately, its .lambda..sub.EM is too short to avoid significant interference from background interference in biological samples, there is no means for converting it to a direct label, it cannot be excited efficiently by Argon 488 line and its efficient excitation with UV light requires expensive quartz optics.
Fluorescein is a widely used fluorophore with a .lambda..sub.EX of 492 nm and a .lambda..sub.EM of 520 nm (Stokes Shift of 28 nm). Hemmila, supra, p112. Fluorescein has a high quantum yield and is excited efficiently by Argon laser, but the Stokes Shift is quite small. Further the .lambda..sub.EM is too short to avoid some background interference in typical biological samples and there no functionality on the fluorescein molecule that can be used to solubilize galactoside and similar substrate derivatives to render such derivatives really of practical utility. Finally, fluorescein is known to be quite photo-labile, making it a difficult fluorophore to use in many applications.
Rhodamine (as TRITC) is a widely used fluorophore with a .lambda..sub.EX of 540 nm and a .lambda..sub.EM of 575 nm (Stokes Shift of 35 nm). Hemmila, supra. Its emission wavelength is longer than most background sources, but its Stokes Shift also is quite small. Further, it has no functional group that can be denivatized for enzyme substrate preparation.
Cy2, a cyanine dye introduced in recent years (Southwick et al., Cytometry II:418, 1990) has a .lambda..sub.EX of 489 nm and a .lambda..sub.EM of 506 nm (Stokes Shift 18 nm). The .lambda..sub.EM of this fluorophore is optimal for use with the Argon laser, but the Stokes Shift is extremely small. Further, Cy2 has no moiety for converting the fluorophore to an enzyme substrate.
2'-(2-Benzothiazolyl)-6'-hydroxy-benzothiazole (BBT) has a .lambda..sub.EX of 419 nm and a .lambda..sub.EM of 561 nm with the Stokes Shift being 142 nm (U.S. Pat. No. 5,424,440, hereby incorporated by reference). BBT, a dibenzothiazole derivative, has several desirable properties as a fluorophore. It has an exceptionally large Stokes shift (142 nm) and has a moiety (phenolic hydroxyl group) that serves as a handle for conversion to enzyme substrates, such as the phosphate (AttoPhos.RTM., JBL Scientific, Inc., San Luis Obispo, Calif.). However, this material has a number of significant shortcomings. First, the .lambda..sub.EX is too short to be maximally excited by an Argon 488 nm laser. Second, the .lambda..sub.EM is long enough (561 nm) to be above the most intense background fluorescence present in serum samples but is still too short to be above some of the weaker background interference in a band from 540 to 580 nm, as described in Hemmila (supra, p.65). Third, derivatives of BBT that could serve as substrates specific for several important enzymes, such as galactosidase, esterases, and lipases are not of practical value because of quite limited solubility of the resulting compounds (e.g., much less than 1 mM solubility for the galactoside of BBT, making zero order kinetics impossible to achieve over a wide range of concentrations of enzyme). Fourth, BBT is a suitable fluorophore for use as an alkaline phosphatase derivative (AttoPhos.RTM.) since its pKa is 8.5 and is fully ionized and therefore fully fluorescent at the pH typically employed for ALP assay (pH 10.3). However, the assay for many enzymes, such as beta-D-galactosidase, esterases and lipases, is preferably performed in the pH range of 6.0 to 8.0. In this pH range BBT is only partially ionized and, therefore, much less intensely fluorescent. This is a further barrier to the use of BBT in this type of application. Fifth, there is no means by which BBT can be used as a direct label, since the only functional group available is the phenolic hydroxyl moiety, the derivatization of which prevents the fluorescence at 560 nm. Finally, BBT is a fluorophore introduced with the choice of only one wavelength and no indication of how additional wavelengths might be achieved. There are several interesting applications, involving simultaneous detection of two or more targets, which require the availability of two or more fluorophores.
2-Phenyl-6-hydroxybenzoxazole (Habenstein, U.S. Pat. No. 5,585,247) has a .lambda..sub.EX of 370 nm and a .lambda..sub.EM of 460 nm with Stokes Shift being 110 nm. This is a benzoxazole derivative that also has a larger than usual Stokes shift (110 nm), but it is optimally excited by UV light, not an Argon laser. The emission is not long enough to avoid the interfering background fluorescence of typical biological samples. Further, there are no means indicated to affect solubility nor the dependence of fluorescence on pH. Derivatives prepared from this fluorophore thus have limited practical usefulness as substrates for such applications as measurement of lipase, esterase and glycolytic enzymes. Further, there is no means by which this fluorophore might be utilized as a direct label.